WO2003035669A1 - Lipides cationiques amphiphiles derives du cholesterol - Google Patents

Lipides cationiques amphiphiles derives du cholesterol Download PDF

Info

Publication number
WO2003035669A1
WO2003035669A1 PCT/US2001/032399 US0132399W WO03035669A1 WO 2003035669 A1 WO2003035669 A1 WO 2003035669A1 US 0132399 W US0132399 W US 0132399W WO 03035669 A1 WO03035669 A1 WO 03035669A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
group
chol
formula
cholesterol
Prior art date
Application number
PCT/US2001/032399
Other languages
English (en)
Inventor
Szepsel Gerszberg
Daniel Alonso
Original Assignee
Sterrenbeld Biotechnologie North America, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sterrenbeld Biotechnologie North America, Inc. filed Critical Sterrenbeld Biotechnologie North America, Inc.
Priority to PCT/US2001/032399 priority Critical patent/WO2003035669A1/fr
Priority to MXPA04003761A priority patent/MXPA04003761A/es
Publication of WO2003035669A1 publication Critical patent/WO2003035669A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane

Definitions

  • This invention relates to the description and synthesis of lipids.
  • the cationic lipids are characterized by having a hydrophilic domain and a hydrophobic domain. These domains are formed by long carbon chains. The most active molecules are positively charged and are associated to laminar structures containing multiple positive charges on their surfaces.
  • cationic lipids are known in the art. According to their structure, these groups are classified into cholesterol or glycerin derivatives. The glycerin group may be divided further in diacylglyceryl or dietherglyceryl sub-groups.
  • amphiphilic cationic lipids are used currently in gene therapy to transfer DNA and RNA to cultured animal cells. These compounds facilitate an efficient non-viral transference (transfection, lipofection) of nucleic acids into cells.
  • the amphiphilic cationic lipids are lipophilic molecules charged positively that form complexes easily with DNA and other anionic polynucleotides.
  • the cationic lipids known in the art require mixing with variable amounts of neutral, zwitterionic lipids such as DOPE (dioleoylphosphatidylethanolamine) to stabilize their aqueous emulsion and optimize transfection.
  • DOPE dioleoylphosphatidylethanolamine
  • the mixture of the cationic lipid with a neutral phospholipid increases its activity.
  • This combination results in cationic liposomes which are usually more efficient in cell transfection than its individual components.
  • These liposomes are characterized by preserving all the properties of both amphipathic subrogates of the precursor cationic lipid.
  • Cationic liposomes have a net polycationic surface capable of strong interaction with the polyanionic phosphate backbone of polynucleotides. Such interaction occurs spontaneously.
  • lipoplex- The complex formed by the polynucleotide, the cationic lipid and the neutral phospholipid -referred to as lipoplex- interacts strongly with the negative charge prevailing in the cell surface. This phenomenon favors the lipoplex-plasmatic membrane fusion and the effective transference of the polynucleotide into the cellular cytoplasm.
  • Amphiphilic cationic lipids are characterized by containing an amino group, a spacer arm, a linking bond and a lipophilic group.
  • the amino group is charged positively at neutral pH.
  • the amino group may be primary, secondary, tertiary or quaternary.
  • the lipophilic group is a hydrophobic unit or portion that allows the insertion of the cationic amphiphile into the membrane of the cell or liposome.
  • the lipophilic group serves as an anchor for the cationic ammonium group to attach to the surface of a cell or liposome.
  • the spacer group is usually hydrophilic and has a variable length and composition.
  • the function of the spacer group is to connect the amino group to the lipophilic group by means of a chemical bond. It is preferable that the chemical bond be hydrolizable, i.e. esters, amides, carbonate or carbamate.
  • the molecule cationic region and the hydrophobic lipophilic region of the cationic lipid are biocompatible. That is, the hydrophobic and cationic units either occurs naturally or is well tolerated by cells.
  • the connecting union of cationic lipids is biodegradable.
  • a biodegradable union is that which degrades or hydrolyzes under certain conditions or during normal processes in cells or organisms.
  • the present patent claims amphiphilic cationic lipids not presently known in the art.
  • the present patent also describes the synthesis of said amphiphilic cationic lipids.
  • the claimed compounds have the following advantages, among others: low toxicity, biocompatibility of the hydrophilic and hydrophobic domains and biodegradability of its connecting bond. Because of these characteristics, the claimed lipids may be used for in vivo, in vitro and ex vivo gene transfer and for medical treatments based on gene therapy.
  • lipids claimed in the present patent have the additional advantage of preserving their chemical integrity in solution while hydrolyzing when in contact with tissue. This characteristic makes the claimed lipids more stable and extends their half-life without altering their therapeutical utility.
  • Another advantage of the lipids claimed herein is their rigid planar hydrophobic backbone that does not alter the hydrocarbonated packing in the bilayer membrane.
  • the claimed lipids include also a positively charged tertiary or quaternary amine group that enhances further the bilayer stability.
  • the present invention claims amphiphilic cationic lipids derived from cholesterol characterized by the following chemical structure: 1) an hydrophilic domain which includes a dimethylamine group, 2) a spacer group (R ⁇ ) which includes at least 2 carbon atoms linked to the hydrophilic domain, 3) one linking bond (L) that includes a carboxyl ester, carbamate or carbonate group linked to the spacer group and 4) one lipophilic group derived from cholesterol (Choi) attached to the linking bond.
  • the hydrophilic domain of the claimed amphiphilic cationic lipids is a tertiary dimethylamine group.
  • the present invention has the following overall structure:
  • the hydrophilic domain of the claimed amphiphilic cationic lipids is a quaternary dimethylamine group.
  • the quaternary dimethylamine group is formed by binding a tertiary dimethylamine group to a 1,2-halohydrin or a 1,3-halohydrin.
  • the tertiary dimethylamine group is linked to a 1 ,2-halohydrin; more preferably, to 2-hydroxyethyl.
  • the present invention has the following overall chemical structure:
  • Ri represents a spacer group composed by at least two carbon atoms
  • R 2 represents either a 2-hidroxyalkyl group or 3-hydroxyalkyl group
  • L represents a linking bond between a lipophilic group (Choi) and the R ⁇ chain, and L includes a carboxyl ester, carbamate or carbonate group, Choi represents the cholesterol tetracyclic core without the ⁇ hydroxyl at C-3 substituted in said position by a linking bond (L), and
  • X represents an anion
  • the linking bond includes a carbamate group and has the following overall chemical structure:
  • the linking bond includes a carbonate group and has the following overall chemical structure:
  • the linking bond includes a carboxyl ester group and has the following overall chemical structure:
  • the Ri spacer group may be an aliphatic chain or contain an aromatic radical.
  • the aliphatic chain may be linear or branched. In one embodiment of the present invention, the aliphatic chain is linear and saturated. In another embodiment of the present invention, the aliphatic chain is unsaturated.
  • the spacer group contains a heteroatom, i.e. nitrogen or oxygen. In such cases, the heteroatom forms an additional ester, amide, amino or ether group.
  • the aromatic radical is a phenyl group.
  • the spacer group has more than 2 carbon atoms. More preferably, the spacer group has less than 10 carbon atoms.
  • the quaternary dimethylamine group forms bonds with negatively charged ions.
  • anions that may form the quaternary salts claimed in the present invention are chloride, bromide, iodine, acetate, sulfate, bisulfate, trifluoroacetate, trifluoromethanesulfonate and monophosphate.
  • the anion linked to the quaternary dimethylamine group is bromide.
  • Figure 1 illustrates the synthesis of HDC-Chol (11) and represents its chemical structure.
  • Figure 2 illustrates the synthesis of HCC-Chol (IN) and represents its chemical structure.
  • Figure 3 illustrates the syntheses of AB-Chol (N) and HAB-Chol (NI) and represents their chemical structures.
  • Figure 4 illustrates the synthesis of HDS-Chol (LX) and represents its chemical structure.
  • Figure 5 illustrates the syntheses of CS-Chol (X) and HCS-Chol (XI) and represents their chemical structures.
  • DC-Choi (I) was prepared from cholesteryl chloroformate and ⁇ , ⁇ - dimethylethylenediamine applying techniques known in the art. See Gao et al,
  • CC-Chol (HI) was obtained by reaction of cholesteryl chloroformate with 2-(dimethylamino)ethanol according to the technique described in WO 98/10649 (Shefter et als).
  • the 2-bromoethanol quaternization was produced according to the Moss technique. See Moss et al., J. Amer. Chem. Soc, 109 (19): 5740-5744 (1987).
  • the melting points were determined with a Mel-Temp II equipment and were uncorrected.
  • the 13 C nuclear magnetic resonance spectra (NMR) were determined with a Brucker MSL 300 spectrophotometer at 75.47 MHz. Chemical shifts were expressed as parts per million (ppm) relative to tetramethylsilane used as internal standard. Deuterated chloroform or deuterated dimethylsulfoxide were used as solvent.
  • the mass spectra (MS) were determined with a Quatro II-Micromass instrument using the FAB mode.
  • the infrared spectra were determined with a FT-IR Perkin Elmer Spectrum BX equipment utilizing a KBr solid dispersion.
  • TLC was performed using silica gel plates 60G.
  • the plates were developed by spraying with sulfuric acid in methanol 10% (v/v) and heating at 110 °C.
  • the quaternary salt thus obtained was purified by column chromatography.
  • the salt was eluted with a CHCl 3 :MeOH (9:1) mixture.
  • the pure fractions obtained were evaporated to dryness. 1.96 g of a yellowish white solid identified as HDC-Chol was thus obtained.
  • the reaction yield was 52.3%. See Fig. 1.
  • reaction yield was 75.5%.
  • the semisolid residue was purified by a silica gel column chromatography to separate the wax-like product according to the following protocol.
  • the semisolid residue was treated with CHC1 and first eluted with a mixture of CHCl 3 :MeOH 3% (v/v) to remove the Rf 0.79 impurity. Then, the eluting mixture was increased with CHCl 3 :MeOH 5% (v/v) and the Rf 0.25 product was eluted.
  • the resulting pure fractions were evaporated to dryness and an oily product was obtained. The oily product solidified later into a yellowish white wax. 10.6 g of a product identified as AB-Chol was thus obtained.
  • the reaction yield was 88.8%. See Fig. 3.
  • reaction mixture was evaporated to dryness and the resulting residue was purified by silica gel column chromatography. A CHCl 3 :MeOH (4:1 v/v) mixture was utilized as eluent. The evaporation to dryness of the product pure fractions yielded a yellowish white wax-like residue. 1.04 g of a product identified as HAB-Chol was thus obtained. The reaction yield was 83.2%. See Fig. 3.
  • reaction mixture was evaporated to dryness.
  • the resulting oily residue was purified using a silica gel column chromatography. A CHCl 3 :MeOH (9:1 v/v) mixture was utilized as eluent. Pure fractions of this product were treated next with methanol. Then, the evaporation to dryness of the product pure fractions yielded a wax-like yellowish white solid. 0.86 g of a product identified as HDS-Chol was thus obtained. The reaction yield was 40.1%. See Fig. 4.
  • reaction mixture was stirred magnetically for 8 hours.
  • a solution of 2.8 ml (27.86 mmol) of 2-(dimethylamino)ethanol in 30 ml of anhydrous dichloromethane was added then drop by drop to the reaction mixture.
  • the reaction mixture was left stirring overnight at room temperature.
  • the reaction mixture was evaporated to dryness.
  • the resulting oily residue is prone to solidify in storage.
  • the residue was treated with acetonitrile (40 ml) and cooled in an ice-water bath. A yellowish white wax-like solid resulted.
  • This solid was centrifuged and rinsed thrice with acetonitrile. The solid was dried then with air. 4.01 g of a product identified as CS-Chol was thus obtained.
  • the reaction yield was 87.5%. See Fig. 5.
  • the reaction mixture was evaporated then to dryness.
  • the resulting oily residue was purified by a silica gel column chromatography according to the following protocol.
  • the oily residue was grown in CHC1 3 .
  • the substrate and the impurity traces were eluted with a CHCl 3 :MeOH (9:1 v/v) mixture.
  • the product of interest was eluted then with a CHCl 3 :MeOH (8:2 v/v) mixture.
  • the evaporation of product pure fractions resulted in a yellowish white wax-like solid. 0.41 g of a product identified as HCS-Chol was thus obtained.
  • the reaction yield was 63.8%. See Fig. 5.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Steroid Compounds (AREA)
  • Medicinal Preparation (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

Abstract

L'invention concerne des lipides cationiques amphiphiles dérivés du cholestérol, qui renferment: 1) un domaine hydrophile comprenant un groupe diméthylamine, 2) un groupe intercalaire comportant au moins deux atomes de carbone reliés au domaine hydrophile, 3) une liaison fonctionnelle comprenant un groupe constitué d'un carboxyl ester, d'un carbamate et d'un carbonate et relié au groupe intercalaire, et 4) un groupe lipophile dérivé du cholestérol se rattachant à la liaison fonctionnelle.
PCT/US2001/032399 2001-10-22 2001-10-22 Lipides cationiques amphiphiles derives du cholesterol WO2003035669A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/US2001/032399 WO2003035669A1 (fr) 2001-10-22 2001-10-22 Lipides cationiques amphiphiles derives du cholesterol
MXPA04003761A MXPA04003761A (es) 2001-10-22 2001-10-22 Lipidos cationicos anfofilos derivados del colesterol.

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US2001/032399 WO2003035669A1 (fr) 2001-10-22 2001-10-22 Lipides cationiques amphiphiles derives du cholesterol

Publications (1)

Publication Number Publication Date
WO2003035669A1 true WO2003035669A1 (fr) 2003-05-01

Family

ID=21742918

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2001/032399 WO2003035669A1 (fr) 2001-10-22 2001-10-22 Lipides cationiques amphiphiles derives du cholesterol

Country Status (2)

Country Link
MX (1) MXPA04003761A (fr)
WO (1) WO2003035669A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007022030A2 (fr) * 2005-08-11 2007-02-22 University Of Massachusetts Procedes et compositions pour l'apport efficace d'agents therapeutiques dans des cellules et des animaux
WO2008155141A2 (fr) * 2007-06-20 2008-12-24 Novosom Ag Nouveaux stérols facultativement cationiques

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5283185A (en) * 1991-08-28 1994-02-01 University Of Tennessee Research Corporation Method for delivering nucleic acids into cells
US5935936A (en) * 1996-06-03 1999-08-10 Genzyme Corporation Compositions comprising cationic amphiphiles and co-lipids for intracellular delivery of therapeutic molecules

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5283185A (en) * 1991-08-28 1994-02-01 University Of Tennessee Research Corporation Method for delivering nucleic acids into cells
US5935936A (en) * 1996-06-03 1999-08-10 Genzyme Corporation Compositions comprising cationic amphiphiles and co-lipids for intracellular delivery of therapeutic molecules

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007022030A2 (fr) * 2005-08-11 2007-02-22 University Of Massachusetts Procedes et compositions pour l'apport efficace d'agents therapeutiques dans des cellules et des animaux
WO2007022030A3 (fr) * 2005-08-11 2007-05-24 Univ Massachusetts Procedes et compositions pour l'apport efficace d'agents therapeutiques dans des cellules et des animaux
WO2008155141A2 (fr) * 2007-06-20 2008-12-24 Novosom Ag Nouveaux stérols facultativement cationiques
WO2008155141A3 (fr) * 2007-06-20 2009-03-19 Novosom Ag Nouveaux stérols facultativement cationiques

Also Published As

Publication number Publication date
MXPA04003761A (es) 2005-04-08

Similar Documents

Publication Publication Date Title
US11679158B2 (en) Biodegradable lipids for the delivery of active agents
JP6226476B2 (ja) カチオン性脂質
US9668973B2 (en) Components for producing amphoteric liposomes
JP5902616B2 (ja) カチオン性脂質
JP5902617B2 (ja) カチオン性脂質
JP4965489B2 (ja) カチオン脂質複合体
JPWO2013089151A1 (ja) カチオン性脂質を含有するドラックデリバリーシステムのための脂質ナノ粒子
US20090215067A1 (en) Novel lipids for transfection of nucleic acids
CN111417621A (zh) 阳离子脂质
EP1041976A1 (fr) Oligomeres polyamidiques
US6489495B2 (en) Cationic amphiphiles for liposomal gene transfer
EP1666486A1 (fr) Compose modifie a l'aide d'un derive de glycerol
WO2003035669A1 (fr) Lipides cationiques amphiphiles derives du cholesterol
ITMI961684A1 (it) Chelati paramagnetici ad alta relassivita' in siero
JP6495408B2 (ja) カチオン性脂質
EP3153172A1 (fr) Composition pharmaceutique à base d'arni réprimant la transcription du gène ckap5
WO2000012454A1 (fr) Nouveaux lipides cationiques
JPH0426699A (ja) アミノ酸誘導体及びその製造法
US5399590A (en) Quaternary ammonium salts and pharmaceutical compositions containing them
CN113683769B (zh) 一种细胞内吞释放响应的化合物及其应用
CN117919199A (zh) 空白脂质纳米颗粒在制备体内递送产品中的应用
CN117942406A (zh) 预制载体在制备用于免疫细胞和干细胞的体外基因递送的产品中的应用
KR20040025178A (ko) 양이온성 계면활성제의 제조방법

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP